3 research outputs found
Fast cation-exchange separation of proteins in a plastic microcapillary disc
A novel disposable adsorbent material for fast cation-exchange separation of proteins was developed based on plastic microcapillary films (MCFs). A MCF containing 19 parallel microcapillaries, each with a mean internal diameter of 142μm, was prepared using a melt extrusion process from an ethylene-vinyl alcohol copolymer (EVOH). The MCF was surface functionalised to produce a cation-exchange adsorbent (herein referred as MCF-EVOH-SP). The dynamic binding capacity of the new MCF-EVOH-SP material was experimentally determined by frontal analysis using pure protein solutions in a standard liquid chromatography instrument for a range of superficial flow velocities, u =5.5-27.7cms . The mean dynamic binding capacity for hen-egg lysozyme was found to be approximately 100μg for a 5m length film, giving a ligand binding density of 413ngcm . The dynamic binding capacity did not vary significantly over the range of u tested. The application of this novel material to subtractive chromatography was demonstrated for anionic BSA and cationic lysozyme at pH 7.2. The chromatographic separation of two cationic proteins, lysozyme and cytochrome-c, was also performed with a view to applying this technology to the analysis or purification of proteins. Future applications might include separation based on anion exchange and other modes of adsorption. © 2011 Elsevier B.V
Amphipathic polymer-mediated uptake of trehalose for dimethyl sulfoxide-free human cell cryopreservation
For stem cell therapy to become a routine reality, one of the major challenges to overcome is their storage and transportation. Currently this is achieved by cryopreserving cells utilising the cryoprotectant dimethyl sulfoxide (MeSO). MeSO is toxic to cells, leads to loss of cell functionality, and can produce severe side effects in patients. Potentially, cells could be frozen using the cryoprotectant trehalose if it could be delivered into the cells at a sufficient concentration. The novel amphipathic membrane permeabilising agent PP-50 has previously been shown to enhance trehalose uptake by erythrocytes, resulting in increased cryosurvival. Here, this work was extended to the nucleated human cell line SAOS-2. Using the optimum PP-50 concentration and media osmolarity, cell viability post-thaw was 60±2%. In addition, the number of metabolically active cells 24h post-thaw, normalised to that before freezing, was found to be between 103±4% and 91±5%. This was found to be comparable to cells frozen using MeSO. Although reduced (by 22±2%, p=0.09), the doubling time was found not to be statistically different to the non-frozen control. This was in contrast to cells frozen using MeSO, where the doubling time was significantly reduced (by 41±4%, p=0.004). PP-50 mediated trehalose delivery into cells could represent an alternative cryopreservation protocol, suitable for research and therapeutic applications. © 2013 The Authors
Amphipathic polymer-mediated uptake of trehalose for dimethyl sulfoxide-free human cell cryopreservation
For stem cell therapy to become a routine reality, one of the major challenges to overcome is their storage and transportation. Currently this is achieved by cryopreserving cells utilising the cryoprotectant dimethyl sulfoxide (MeSO). MeSO is toxic to cells, leads to loss of cell functionality, and can produce severe side effects in patients. Potentially, cells could be frozen using the cryoprotectant trehalose if it could be delivered into the cells at a sufficient concentration. The novel amphipathic membrane permeabilising agent PP-50 has previously been shown to enhance trehalose uptake by erythrocytes, resulting in increased cryosurvival. Here, this work was extended to the nucleated human cell line SAOS-2. Using the optimum PP-50 concentration and media osmolarity, cell viability post-thaw was 60±2%. In addition, the number of metabolically active cells 24h post-thaw, normalised to that before freezing, was found to be between 103±4% and 91±5%. This was found to be comparable to cells frozen using MeSO. Although reduced (by 22±2%, p=0.09), the doubling time was found not to be statistically different to the non-frozen control. This was in contrast to cells frozen using MeSO, where the doubling time was significantly reduced (by 41±4%, p=0.004). PP-50 mediated trehalose delivery into cells could represent an alternative cryopreservation protocol, suitable for research and therapeutic applications. © 2013 The Authors